The activity and cell surface density of Kir 1.1 (ROMK) channels are exquisitely controlled to regulate potassium secretion and sodium reabsorption in the kidney. Loss-of-function mutations lead to Bartter's syndrome, a hereditary salt-losing nephrology. Here we propose to elucidate key molecular mechanisms that operate to regulate the activity and surface density of these channels, building on our recent our discoveries. A stepwise multidisciplinary approach, combining molecular genetics, cellular biology, electrophysiology and transgenics, will be employed to answer the following questions about the regulation of channel gating and surface density: 1) how does the cytoplasmic N-terminal/COOH-terminal interface control channel gating? Our work on molecular basis of channel gating defects in Bartter's syndrome suggests that a key inter-subunit interaction site may control the energetics of channel opening. This Aim is designed to critically explore this idea, testing whether the interaction site controls pH-dependent channel gating, as conferred by titration of an N-terminal pH sensor. 2) How is the ROMK channel processed in the biosynthetic pathway? This Aim is designed to elucidate the structural basis and consequence of ER retention, providing a context to understand how phosphorylation by PKA/SGK and PDZ interactions regulate ROMK expression on the plasma membrane. 3) What is the molecular mechanism by which PKA/SGK phosphorylation regulates cell surface expression of ROMK? This Aim is designed to test the hypothesis that phosphorylation of serine 44 controls anterograde traffic from the ER/Golgi by abrogating an "ER retention" signal. 4) How does NHERF-2 interaction with ROMK regulate cell surface expression? This Aim will test the hypothesis that PDZ-interactions stabilize ROMK expression on the plasmalemma, building on our discovery that the PDZ protein, NHERF-2, coordinates the assembly of a multimeric protein complex around ROMK and facilitates channel expression on the plasmalemma. 5) Does NHERF-2 interaction regulate ROMK expression in the CCD during potassium adaptation? In this Aim, we will use wild type and NHERF-2 knock-out mice to test whether NHERF-2 interaction with ROMK underpins physiological regulation of ROMK in potassium homeostasis. These studies represent a timely and important extension of the principal investigator's work, and should ultimately provide considerable insight into the basis of renal K handling and K homeostasis in health and disease.